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It is held as a paradigm that ribosomally synthesized peptides and proteins contain only L-amino acids. We demonstrate a ribosomal origin of the marine sponge—derived polytheonamides, exceptionally potent, giant natural-product toxins. Isolation of the biosynthetic genes from the sponge metagenome revealed a bacterial gene architecture. Only six candidate enzymes were identified for 48 posttrarelational modifications, including 18 epimerizations and 17 methylations of nonactivated carbon centers. Three enzymes were functionally validated, which showed that a radical S-adenosylmethionine enzyme is responsible for the unidirectional epimerization of multiple and different amino acids. Collectively, these complex alterations create toxins that function as unimolecular minimalistic ion channels with near-femtomolar activity. This study broadens the biosynthetic scope of ribosomal systems and creates new opportunities for peptide and protein bioengineering.

Investigations into the biosynthetic pathways of three families of actin-targeting macrolides lead to insights into their convergent or combinatorial evolution, along with the identification of the first free-living bacterial source of macroalga-derived luminaolides. Actin-targeting macrolides comprise a large, structurally diverse group of cytotoxins isolated from remarkably dissimilar micro- and macroorganisms. In spite of their disparate origins and structures, many of these compounds bind actin at the same site and exhibit structural relationships reminiscent of modular, combinatorial drug libraries. Here we investigate biosynthesis and evolution of three compound groups: misakinolides, scytophycin-type compounds and luminaolides. For misakinolides from the sponge Theonella swinhoei WA, our data suggest production by an uncultivated 'Entotheonella' symbiont, further supporting the relevance of these bacteria as sources of bioactive polyketides and peptides in sponges. Insights into misakinolide biosynthesis permitted targeted genome mining for other members, providing a cyanobacterial luminaolide producer as the first cultivated source for this dimeric compound family. The data indicate that this polyketide family is bacteria-derived and that the unusual macrolide diversity is the result of combinatorial pathway modularity for some compounds and of convergent evolution for others.

This research work aimed at investigating the chemistry and biosynthetic potential of ascidian-associated symbionts that originated from Manado Bay, North Sulawesi. We initially enriched the symbiotic cells associated with the Manadonese ascidian Lissoclinum patella . Subsequently we identified the presence of Prochloron didemni in both unenriched and salt-enriched samples by examining the 16S rRNA gene and the chlorophyll A oxygenase (CAO) gene. Investigation of the secondary metabolites by HPLC, LCMS/MS, and NMR showed the presence of ulithiacyclamide along with patellamide E in the unenriched symbiotic cells. Interestingly, ulithiacyclamide was detected in the enriched cells from the same Ascidian specimen. Molecular docking showed the high binding affinity of both compounds to estrogen receptor beta (ER-β) protein. This is a subtype of the nuclear receptor superfamily expressed abnormally in ovarian cancer cells. Furthermore, we isolated a patE gene variant encoding for a precursor with patellamide E (VTVCITFC) and ulithiacyclamide (CTLCCTLC) core peptides from the enriched cells. This represents a new core peptide combination. The outcome of this work provides a basis for producing useful cyclic peptides, in sustainable way, through symbiont cultivation. This could become a platform for bioengineering to generate diverse compound analogues.

Cultivated bacteria such as actinomycetes are a highly useful source of biomedically important natural products. However, such ‘talented’ producers represent only a minute fraction of the entire, mostly uncultivated, prokaryotic diversity. The uncultured majority is generally perceived as a large, untapped resource of new drug candidates, but so far it is unknown whether taxa containing talented bacteria indeed exist. Here we report the single-cell- and metagenomics-based discovery of such producers. Two phylotypes of the candidate genus ‘ Entotheonella ’ with genomes of greater than 9 megabases and multiple, distinct biosynthetic gene clusters co-inhabit the chemically and microbially rich marine sponge Theonella swinhoei . Almost all bioactive polyketides and peptides known from this animal were attributed to a single phylotype. ‘ Entotheonella ’ spp. are widely distributed in sponges and belong to an environmental taxon proposed here as candidate phylum ‘Tectomicrobia’. The pronounced bioactivities and chemical uniqueness of ‘ Entotheonella ’ compounds provide significant opportunities for ecological studies and drug discovery. Single-cell- and metagenomics-based study reveals two members of the candidate genus ‘ Entotheonella ’, symbionts of the marine sponge Theonella swinhoei ; distinct biosynthetic gene clusters that account for most of the bioactive polyketides and peptides known from T. swinhoei are shown to be attributable to a single member of the T. swinhoei Y microbiome. Chemical diversity in marine microbes Almost all drugs and drug candidates from bacteria are produced by a few groups of metabolically rich organisms. That leaves the unculturable — or uncultivated — microbial majority as a largely untapped resource. Here Jörn Piel and colleagues report the use of single-cell and metagenomic analysis to identify two potential 'environmental factories', both members of the candidate genus Entotheonella and symbionts of the chemically rich marine sponge Theonella swinhoei . Importantly they find that the genomes of both microbes encode multiple distinct biosynthetic gene clusters that together account for most of the bioactive polyketides and peptides previously thought to be produced by the sponge host. This discovery identifies Entotheonella and members of the newly proposed phylum Tectomicrobia as a 'biochemically talented' phylum on a par with the actinomycetes.

There has been emerging evidence that the bacteria associated with marine sponges are the key producers of many complex bioactive compounds. The as-yet uncultured candidate bacterial genus “Candidatus Entotheonella” of the marine sponge Theonella swinhoei from Japan have recently been recognized as the source of numerous pharmacologically relevant polyketides and modified peptides, as previously reported by the Piel group (Wilson et al. 2014). This work reported the presence of “Candidatus Entotheonella sp.” in the highly complex microbiome of an Indonesian marine sponge from Kapoposang Island, South Sulawesi. We further identified the Kapoposang sponge specimen used in this work as Rhabdastrella sp. based on the integrated morphological, histological, and cytochrome oxidase subunit I (COI) gene analyses. To detect the polyketide biosynthetic machinery called type I polyketide synthase (PKS) in this Indonesian Rhabdastrella sp., we amplified and cloned the ketosynthase-encoding DNA regions of approximately 700 bp from the uncultured sponge's microbiome. Further sequencing and analysis of several randomly chosen clones indicated that all of them are mostly likely involved in the biosynthesis of methyl-branched fatty acids. However, employing a PKS-targeting primer designed in this work led to the isolation of four positive clones. BlastX search and subsequent phylogenetic analysis showed that one of the positive clones, designed as RGK32, displayed high homology with ketosynthase domains of many type I PKS systems and may belong to the subclass cis-AT PKS group.

Nature 506, 58–62 (2014); doi:10.1038/nature12959 One of the accession numbers for this Article was listed as AZHXW01000000 instead of AZHX01000000. It has been corrected in the online versions of the paper.